Humans are exposed to a large number of chemicals and other environmental factors that interact in a variety of ways ultimately to cause cancer. Cells undergo a progressive series of alterations as they change from the normal to the neoplastically transformed state. If this progression can be slowed, the time required for tumor development may be longer than the lifespan of the animal; hence no tumors will form. This progression is affected by substances termed tumor promoters. Promoters influence the expression of changes caused by initiators (carcinogens, oncogens, mutagens). This progression has been studied extensively in mouse skin. The most frequently used promoters have been the phorbol esters. Since tumor promoters are tissue specific, it is necessary to study this process in other tissues using other promoters. The objective of this research is to develop a new in vitro model for tumor promotion. Cultured hepatocytes were chosen because 1) hepatocytes are promoted in vivo by nonphorbol ester tumor promoters such as phenobarbital, and 2) many biochemical changes occur in hepatocellular carcinogenesis in vivo, some of which are sensitive to promotion in vivo. In this model, cells are altered by the carcinogen in vivo, and then promoted in vitro. Biochemical and biological changes induced by liver tumor promoters will be evaluated by several in vitro markers. This model will be useful in detecting tumor promoters and studying their mechanism of action. It can be used to determine whether additional mutational events are involved, to detect other liver tumor promoters, to evaluate the changes occurring in the cells during tumor progression, to determine the effects of tumor promoters on this progression, and to detect substances that interfere with this progression. This should lead to an understanding of the mechanisms involved in promotion and progression and to means of interfering with these processes and preventing the development of tumors.